SPCC613.01 Antibody

What is SPCC613.01 Antibody and what organism does it target?

SPCC613.01 Antibody is a research-grade antibody designed to recognize and bind to the SPCC613.01 protein in Schizosaccharomyces pombe (fission yeast). This antibody belongs to a class of reagents developed for detecting specific proteins in S. pombe, similar to other custom antibodies like those with SPAC prefixes (e.g., SPAC1039.04, SPAC821.13c) . The nomenclature indicates that the target protein is encoded by a gene located on chromosome 3 of S. pombe, as the "C" in SPCC typically designates this chromosome. This antibody serves as a valuable tool for studying protein localization, expression, and function in this model organism.

What are the primary applications for SPCC613.01 Antibody in research?

SPCC613.01 Antibody can be employed in multiple experimental techniques commonly used in molecular and cellular biology research. The primary applications include:

• Western blotting - For detecting the presence and quantity of the target protein in cell lysates

• Immunocytochemistry (ICC) - For visualizing protein localization within cells

• Immunohistochemistry (IHC) - For examining protein expression in tissues

• Flow cytometry - For quantitative analysis of protein expression in cell populations

• Immunoprecipitation (IP) - For isolating protein complexes containing the target protein

As with other research antibodies, validation across these specific applications is essential, as an antibody that performs well in one application may not necessarily work optimally in others .

How should SPCC613.01 Antibody be validated before experimental use?

Proper validation of SPCC613.01 Antibody is crucial to ensure experimental reliability. A comprehensive validation approach should include:

• Specificity testing: Verify target specificity using knockout/knockdown controls or comparing wild-type versus mutant strains lacking the target protein .

• Application-specific validation: Test the antibody in each intended application (western blot, IHC, flow cytometry) following standardized protocols .

• Positive and negative controls: Include appropriate controls in each experiment to confirm antibody performance .

• Cross-reactivity assessment: Test for potential cross-reactivity with homologous proteins, particularly important in S. pombe research .

• Reproducibility testing: Ensure consistent performance across multiple experiments and batches .

As noted by experts in antibody validation: "Antibodies should be validated for every application in which they will be used, with each validation process adhering to a well-defined and reproducible protocol" .

How should I design flow cytometry experiments using SPCC613.01 Antibody?

When designing flow cytometry experiments with SPCC613.01 Antibody, follow these methodological guidelines:

• Sample preparation: Optimize fixation and permeabilization protocols specifically for S. pombe cells, as these steps significantly impact antibody accessibility to intracellular targets .

• Controls: Include multiple control types:

  • Unstained cells (autofluorescence control)

  • Isotype controls (non-specific binding assessment)

  • Secondary antibody-only controls (if using indirect staining)

  • Positive and negative biological controls

• Titration: Determine optimal antibody concentration through titration experiments to achieve maximum signal-to-noise ratio .

• Panel design: When using SPCC613.01 Antibody in multicolor panels, carefully select fluorophores to minimize spectral overlap and validate the entire panel once individual antibodies have been validated .

• Gating strategy: Develop a consistent gating strategy based on known biological parameters of S. pombe cells.

As noted by flow cytometry experts: "When validating antibodies for flow cytometry, the degree of difficulty often depends on the target location... flow cytometry does not provide high-resolution subcellular localization information" . Consider this limitation when interpreting results.

What are the recommended protocols for immunohistochemistry with SPCC613.01 Antibody?

For optimal immunohistochemistry results using SPCC613.01 Antibody, consider this methodological approach:

• Sample preparation:

  • Fix S. pombe cells in 4% paraformaldehyde

  • Process and embed samples in paraffin or prepare frozen sections

  • Perform antigen retrieval if necessary (typically heat-induced epitope retrieval in citrate buffer pH 6.0)

• Staining protocol:

  • Block with appropriate serum (typically 5-10% normal serum)

  • Apply optimized dilution of SPCC613.01 Antibody (determine through titration)

  • Incubate at 4°C overnight or at room temperature for 1-2 hours

  • Use appropriate detection system (e.g., polymer-based detection system)

  • Counterstain, dehydrate, and mount

• Controls and validation:

  • Include positive and negative controls in each run

  • Consider using automated staining platforms for consistency

The evidence from similar antibody validation shows that automated systems like "Ventana Benchmark GX" or "Dako Autostainer Link 48" can enhance reproducibility for IHC applications .

What factors affect SPCC613.01 Antibody performance in Western blotting?

Several critical factors influence the performance of SPCC613.01 Antibody in Western blotting:

• Sample preparation:

  • Proper lysis buffer selection for S. pombe cells

  • Effective protease inhibitors to prevent degradation

  • Appropriate protein denaturation conditions

• Gel electrophoresis conditions:

  • Optimal percentage of polyacrylamide gel based on target protein size

  • Running conditions (voltage, time)

• Transfer parameters:

  • Transfer buffer composition

  • Transfer time and voltage

  • Membrane type (PVDF vs. nitrocellulose)

• Antibody conditions:

  • Primary antibody dilution (typically 1:500 to 1:2000)

  • Incubation time and temperature

  • Washing stringency

• Detection system:

  • Secondary antibody selection

  • Signal development method (chemiluminescence, fluorescence)

Evidence suggests that "top cited clones do not always perform the best" in Western blotting, highlighting the importance of empirical validation rather than relying solely on citation metrics .

How can I use SPCC613.01 Antibody to study protein-protein interactions in S. pombe?

For investigating protein-protein interactions involving the SPCC613.01 protein, consider these advanced methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Lyse S. pombe cells under non-denaturing conditions

  • Pre-clear lysate with protein A/G beads

  • Immunoprecipitate with SPCC613.01 Antibody

  • Analyze precipitated complexes by mass spectrometry or Western blotting with antibodies against suspected interaction partners

• Proximity ligation assay (PLA):

  • Fix and permeabilize S. pombe cells

  • Incubate with SPCC613.01 Antibody and antibody against potential interacting protein

  • Use species-specific PLA probes

  • Perform ligation and amplification

  • Analyze fluorescent signals indicating proximity (<40 nm)

• Immunofluorescence co-localization:

  • Double immunostaining with SPCC613.01 Antibody and antibodies against potential interacting proteins

  • Use spectrally distinct fluorophores

  • Analyze co-localization using confocal microscopy and quantitative image analysis

When interpreting results, consider that "for techniques such as Western blot, which rely on samples pooled from many different cell types, flow cytometry provides analysis of distinct cellular populations, meaning selecting appropriate cell types for validation purposes is key" .

What are the best practices for resolving discrepancies in results between different immunoassays using SPCC613.01 Antibody?

When faced with discrepancies in results between different immunoassays using SPCC613.01 Antibody, follow this systematic troubleshooting approach:

• Verify antibody specificity:

  • Re-validate the antibody using knockout/knockdown controls

  • Test for cross-reactivity with homologous proteins in S. pombe

  • Confirm target recognition using recombinant protein or peptide competition assays

• Evaluate technical variables:

  • Compare fixation and sample preparation methods between assays

  • Assess the impact of different detection systems

  • Review buffer compositions and incubation conditions

• Consider biological explanations:

  • Protein conformation differences between assays (native vs. denatured)

  • Post-translational modifications affecting epitope accessibility

  • Expression level variations under different experimental conditions

• Perform comparative analysis:

  • Use multiple antibody clones targeting different epitopes of the same protein

  • Compare results from orthogonal techniques (e.g., mass spectrometry)

  • Quantify discrepancies to determine if they are statistically significant

Research demonstrates that "antibody reactivity should be established on a species-by-species basis" and that performance can vary dramatically between applications .

How can I optimize SPCC613.01 Antibody for detecting low-abundance proteins in S. pombe?

Detecting low-abundance proteins presents a significant challenge in antibody-based assays. For optimizing SPCC613.01 Antibody detection of low-abundance targets:

• Sample enrichment strategies:

  • Subcellular fractionation to concentrate compartment-specific proteins

  • Immunoprecipitation prior to analysis to enrich target protein

  • Synchronization of S. pombe cultures to capture peak expression windows

• Signal amplification methods:

  • Tyramide signal amplification (TSA) for immunohistochemistry

  • Enhanced chemiluminescence (ECL) substrates with extended exposure for Western blotting

  • Multi-layer detection systems with secondary and tertiary reagents

• Advanced detection technologies:

  • Super-resolution microscopy for immunofluorescence

  • Sensitive flow cytometry with high-end instruments

  • Digital droplet PCR combined with immunoprecipitation (ChIP-ddPCR)

• Reducing background signals:

  • Optimize blocking conditions (duration, buffer composition)

  • Increase washing stringency

  • Use monovalent Fab fragments to reduce non-specific binding

Evidence suggests that rabbit monoclonal antibodies may offer improved affinity and specificity compared to mouse monoclonals for detecting challenging targets .

What are the most common causes of non-specific binding with SPCC613.01 Antibody and how can they be addressed?

Non-specific binding is a common challenge in antibody-based assays. The primary causes and solutions for SPCC613.01 Antibody include:

• Insufficient blocking:

  • Solution: Optimize blocking buffer (try different blocking agents like BSA, casein, or normal serum)

  • Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Add 0.1-0.3% Triton X-100 or Tween-20 to reduce hydrophobic interactions

• Excessive antibody concentration:

  • Solution: Perform careful titration experiments to determine optimal concentration

  • Use the minimum concentration that gives specific signal

• Cross-reactivity with similar epitopes:

  • Solution: Pre-absorb antibody with recombinant homologous proteins

  • Validate specificity using knockout/knockdown controls

  • Consider testing alternative antibody clones targeting different epitopes

• Sample preparation issues:

  • Solution: Optimize fixation conditions to preserve epitope structure

  • Ensure complete blocking of endogenous enzymes (peroxidases/phosphatases)

  • Include appropriate detergents in wash buffers

• Detection system problems:

  • Solution: Reduce secondary antibody concentration

  • Include carrier proteins (BSA) in secondary antibody dilution

  • Test alternative detection methods

Research shows that "combining recombinant antibody technologies and high validation standards" significantly improves specificity and reduces non-specific binding .

How can I differentiate between true positive signals and artifacts when using SPCC613.01 Antibody?

Distinguishing genuine signals from artifacts requires a methodical approach:

• Use multiple detection methods:

  • Compare results from different techniques (Western blot, IHC, flow cytometry)

  • True positives should be consistent across multiple methods with appropriate controls

• Include comprehensive controls:

  • Genetic controls (knockout/knockdown)

  • Peptide competition assays

  • Isotype controls

  • Secondary antibody-only controls

  • Biological negative controls

• Validate with orthogonal techniques:

  • Confirm protein expression with mRNA analysis (RT-PCR, RNA-seq)

  • Use mass spectrometry to verify protein identity

  • Employ CRISPR-tagged endogenous proteins for localization studies

• Analyze signal characteristics:

  • True signals should have expected subcellular localization

  • Signal intensity should correlate with known biology

  • Pattern should be consistent with the protein's known function

• Perform biological validation:

  • Verify that signals change as expected with relevant biological stimuli

  • Check consistency with published literature on the target protein

As noted by experts: "Validation should aim to reflect the performance of the antibody in alignment with known biology; this ensures an accurate readout and enables unknown biology to be explored" .

What quantitative methods are recommended for analyzing SPCC613.01 protein expression levels?

For quantitative analysis of SPCC613.01 protein expression using the antibody, consider these methodological approaches:

• Western blot quantification:

  • Use housekeeping proteins (e.g., actin, GAPDH) as loading controls

  • Employ digital image analysis software with linear dynamic range

  • Generate standard curves with recombinant protein if absolute quantification is needed

  • Report results as fold-change relative to control samples

• Flow cytometry quantification:

  • Use median fluorescence intensity (MFI) rather than mean values

  • Include quantification beads for standardization between experiments

  • Apply compensation for spectral overlap in multicolor experiments

  • Present data as histograms and MFI values

• Immunofluorescence quantification:

  • Use confocal microscopy with consistent acquisition settings

  • Perform digital image analysis with appropriate thresholding

  • Measure integrated density, mean intensity, or area of positive staining

  • Analyze sufficient cells to achieve statistical significance

• ELISA/quantitative immunoassays:

  • Develop sandwich ELISA with SPCC613.01 Antibody

  • Generate standard curves with recombinant protein

  • Ensure linear range of detection is appropriate for expected concentration

  • Validate reproducibility across multiple plates/runs

How does SPCC613.01 Antibody compare to similar antibodies targeting S. pombe proteins?

When comparing SPCC613.01 Antibody to other S. pombe-specific antibodies, consider the following aspects:

• Target protein characteristics:

  • SPCC613.01 Antibody targets a specific S. pombe protein, similar to other custom antibodies like SPAC1039.04 or SPAC821.13c

  • The specific function and localization of the target protein will influence optimal detection methods

• Antibody format and production:

  • Like other S. pombe antibodies, SPCC613.01 Antibody is typically available in both monoclonal and polyclonal formats

  • Polyclonal antibodies recognize multiple epitopes, beneficial for certain applications but potentially increasing cross-reactivity

  • Monoclonal antibodies provide greater specificity but may be more sensitive to epitope modifications

• Performance characteristics:

  • Validation data should be compared across applications (Western blot, IHC, flow cytometry)

  • Signal strength, background levels, and reproducibility should be evaluated relative to established antibodies

  • Cross-reactivity profiles with other S. pombe proteins may differ between antibodies

• Application suitability:

  • Some antibodies may excel in particular applications while performing poorly in others

  • Performance variation between applications is common and should guide experimental design

Research indicates that "the highest PD-L1 expression on tumor cells occurs in SCC patients and in adenocarcinoma patients without common, druggable genetic abnormalities," highlighting how antibody performance can vary based on biological context .

What emerging technologies are improving the specificity and sensitivity of antibodies like SPCC613.01?

Several cutting-edge technologies are enhancing antibody performance for research applications:

• Recombinant antibody production:

  • Provides batch-to-batch consistency compared to traditional methods

  • Allows for protein engineering to improve affinity and specificity

  • Enables incorporation of detection tags without additional conjugation steps

• Next-generation sequencing (NGS) antibody selection:

  • Facilitates high-throughput screening of antibody libraries

  • Identifies optimal clones with desired binding characteristics

  • Reduces reliance on traditional hybridoma technology

• Phage display antibody discovery:

  • Allows rapid screening of large antibody libraries

  • Identifies antibodies with superior binding properties

  • Enables in vitro selection under controlled conditions

• Advanced validation technologies:

  • CRISPR-Cas9 knockout validation systems

  • Multiplexed epitope mapping

  • Super-resolution microscopy for enhanced localization studies

• Enhanced conjugation chemistry:

  • Site-specific conjugation methods that preserve antibody function

  • Novel fluorophores with improved brightness and photostability

  • Multiplexing capabilities with minimal spectral overlap

As noted in the research: "Combining recombinant antibody technologies and high validation standards" is key to developing superior reagents for research applications .

What are the future directions for SPCC613.01 Antibody applications in S. pombe research?

The future of SPCC613.01 Antibody applications in S. pombe research is likely to expand in several promising directions:

• Systems biology applications:

  • Integration of antibody-based proteomics with transcriptomics and metabolomics

  • Network analysis of protein interactions in S. pombe

  • Computational modeling of cellular processes based on quantitative antibody data

• Single-cell analysis:

  • Mass cytometry (CyTOF) for high-dimensional protein profiling at single-cell resolution

  • Imaging mass cytometry for spatial proteomics in S. pombe

  • Microfluidic approaches for temporal protein dynamics

• Functional genomics integration:

  • Combining CRISPR-Cas9 genome editing with antibody-based protein detection

  • Correlating genetic variants with protein expression and modification patterns

  • Synthetic biology applications in S. pombe using antibody-based feedback systems

• Advanced microscopy techniques:

  • Expansion microscopy for enhanced spatial resolution

  • Light-sheet microscopy for 3D protein localization

  • Live-cell super-resolution imaging for dynamic protein studies

• Therapeutic and biotechnology applications:

  • Using S. pombe as a model system for developing antibody-based therapeutics

  • Leveraging knowledge of S. pombe proteins for industrial biotechnology

  • Engineering S. pombe for bioproduction of therapeutic proteins

Research indicates that "extensive antibody characterisation and validation is key to antibody specificity and performance," suggesting that continued improvements in validation methods will be crucial for advancing these future applications .